Advanced research in proteins and the development of increasingly sophisticated uses of proteins for diagnostic and therapeutic purposes have drawn attention to proteins which are available only in very minute amounts. Such proteins are currently of interest for antibody preparations and vaccine preparations as well as clinical techniques such as screening and diagnostic protocols to monitor the presence of the proteins, the clinical course of the proteins, or the response of a patient to therapy. Tissue-specific, tumor-related and viral antigens are examples of proteins where this need is particularly evident.
The most successful means of isolating these minute amounts are the various types of electrophoresis, such as for example one- and two-dimensional polyacrylamide gel electrophoresis. Electrophoresis produces gel spots or bands containing purified protein that can be visualized by different methods, such as autophoresis, staining, and autoradiography. The bands or spots are often faint, however, due to the small amount of protein contained within them, and the quantities contained within any single excised gel piece are usually too small for further processing or for use in most clinical, diagnostic or therapeutic preparations. As a result, the identification of these proteins by microsequencing techniques is limited.
In addition, researchers are often called upon to sequence proteins present in small amounts in biological material. Investigators have reported that digestion can occur more efficiently and reliably in protocols which are performed in situ (i.e., either in a gel or on a blot), as compared to those in which the protein is electroeluted, then concentrated and digested in solution. In situ digestion protocols however are often successful only when the protein is present in a concentration considerably higher than that of the typical band or spot isolated directly from the biological material.
Nucleic acids present similar problems. Procedures such as cloning genes, preparing probes for northern blot analyses, and performing studies relating to the structure and function of DNA often require higher concentrations of nucleic acids than can be directly obtained from electrophoretic separations.
To achieve these higher concentrations in either proteins or nucleic acids, the solutes must be extracted from several excised gel pieces containing the same protein or nucleic acid. This is typically done by electroelution of the solute from multiple gel pieces into buffer followed by concentration using various methods. The recovery of solutes from this two-step process is usually low, however. Alternatively, the solute can be extracted and electrophoretically transferred to a recipient gel. Typical arrangements for performing such an extraction in a slab gel between a pair of glass plates are described in two disclosures. One of these is Rasmussen, H. H., et al., "Protein-electroblotting and microsequencing in establishing integrated human protein databases" (in Methods in Protein Sequence Analysis, pp. 103-114, Jornvall, H. V., et at., eds., Advances in life Sciences, Birkauser, Denmark, 1991). Rasmussen, et al. disclose the preparation of a well which is wide enough to accommodate a large number of gel pieces and is formed at the top of a slab gel by inserting four spacers a few centimeters into the gel at the center of the top edge of the gel with adjacent side edges of the spacers touching. The two inner spacers are then removed to leave the well laterally bounded by the remaining two spacers. The well and the two remaining spacers occupy a significant part of the gel volume and the method requires the user not only to use additional spacers but also to attach the spacers to the glass plates enclosing the gel and secure them in position. The other disclosure is Lombard-Platet, G., et al., "Funnel-well SDS-PAGE: a rapid technique for obtaining sufficient quantifies of low-abundance proteins for internal sequence analysis," Biotechniques 15:668-672 (1993). Lombard-Platet, et al. use spacers which are specially shaped to fill in the entire volume between the two glass plates except for a single funnel-shaped well. The upper portion of the well is a wide-angle inverted triangle and the lower portion is a straight vertical channel 5 mm in width. Resolving and stacking gels are poured through the triangular section into the vertical channel, leaving the triangular section open. The gel pieces are then placed in the triangular section above the gel, which is then filled with buffer. As the voltage is applied, the proteins migrate downward and are concentrated into the 5 mm channel. This arrangement requires specially constructed spacers and consumes the entire width of the gel.